FfVel1 and FfLae1, components of a velvet-like complex in Fusarium fujikuroi, affect differentiation, secondary metabolism and virulence

Abstract

Besides industrially produced gibberellins (GAs), Fusarium fujikuroi is able to produce additional secondary metabolites such as the pigments bikaverin and neurosporaxanthin and the mycotoxins fumonisins and fusarin C. The global regulation of these biosynthetic pathways is only poorly understood. Recently, the velvet complex containing VeA and several other regulatory proteins was shown to be involved in global regulation of secondary metabolism and differentiation in Aspergillus nidulans. Here, we report on the characterization of two components of the F. fujikuroi velvet-like complex, FfVel1 and FfLae1. The gene encoding this first reported LaeA orthologue outside the class of Eurotiomycetidae is upregulated in ΔFfvel1 microarray-studies and FfLae1 interacts with FfVel1 in the nucleus. Deletion of Ffvel1 and Fflae1 revealed for the first time that velvet can simultaneously act as positive (GAs, fumonisins and fusarin C) and negative (bikaverin) regulator of secondary metabolism, and that both components affect conidiation and virulence of F. fujikuroi. Furthermore, the velvet-like protein FfVel2 revealed similar functions regarding conidiation, secondary metabolism and virulence as FfVel1. Cross-genus complementation studies of velvet complex component mutants between Fusarium, Aspergillus and Penicillium support an ancient origin for this complex, which has undergone a divergence in specific functions mediating development and secondary metabolism.

Figure 1. Multiple sequence alignment of FfVel1 with other characterized VeA homologous sequences

The alignment was generated using ClustalW. Putative nuclear localization signals (NLS) were predicted using WolF PSORT (Horton et al., 2007) and are marked in shades of red. Putative PEST domains were predicted using the EMBOSS program “epestfind” and marked in green. Sequences aligned are: VeA (AAD42946) from A. nidulans, Ve-1 (XP_957154) from N. crassa, AcVeA (CAL68582) from A. chrysogenum, PcVeA (CAP92389) from P. chrysogenum, FvVe1 (ABC02879) from F. verticillioides and FfVel1 (FN548142) from F. fujikuroi.

Figure 2. Phenotypical analysis of Ffvel1 knock-out and addback strains

A Photographs of the F. fujikuroi wild-type strain IMI58289, the Ffvel1 knock-out strain and the addback strains Ffvel1^C and Ffel1^CΔpat4 grown on different solidified media for 10 days under constant dark conditions. V8 = vegetable juice; CM = complete medium; PDA = Potatoe Dextrose Agar; CD = Czapek Dox (for details see Experimental Procedures). B Spores produced by the F. fujikuroi wild type strain IMI58289, the Ffvel1 knock-out strain and the addback strains Ffvel1^C and Ffel1^CΔpat4 grown for 10 days on V8 solidified media under constant light conditions. Experiment was carried out in triplicate; bars show standard deviations. C Photographs of the F. fujikuroi wild type strain IMI58289 and the Ffvel1 knock-out strain crossed with strain MRC1995. Strains were grown for 6 weeks on V8 solidified media as described in Experimental Procedures.

Figure 3. Microarray analysis of the F. fujikuroi wild-type strain IMI58289 compared to a Ffvel1 knock-out strain

A Venn Diagram of genes upregulated in a Ffvel1 knock-out mutant compared to the WT at 24 h, 72 h and 120 h. Only genes with at least 2 fold upregulation in both replicate hybridizations as compared between strains or time points, were considered to be differentially regulated. B Venn Diagram of genes downregulated in a Ffvel1 knock-out mutant compared to the WT at 24 h, 72 h and 120 h. Only genes with at least 2 fold downregulation in both replicate hybridizations as compared between strains or time points, were considered to be differentially regulated. C Pie charts representing classification of protein sequences corresponding to differentially regulated oligo sets using the MIPS functional database catalogue (for details see Experimental Procedures). D Heat map of genes encoding for proteins involved in secondary metabolism or development as classified by the MIPS functional database catalogue. Each colored square represents the mean log2-fold change of both replicate hybridizations at the given time point (green: up-regulation in the Ffvel1 knock-out mutant compared to the wild type; red: down-regulation in the Ffvel1 knock-out mutant compared to the wild type).

Figure 4. Gibberellin and bikaverin analysis in the Ffvel1 knock-out and overexpression strain OE::Ffvel1 compared to the F. fujikuroi wild type IMI58289

A Photographs of the F. fujikuroi wild-type strain IMI58289, the Ffvel1 knock-out strain and the overexpression strain OE::Ffvel1 grown in 10 % ICI cultures for 3 days. B Expression of selected gibberellin and bikaverin genes in the wild type, the Ffvel1 knockout and the OE::Ffvel1 overexpression mutant. The strains were grown for the indicated time in 10 % ICI medium. The northern blot was hybridized with the indicated probes. 28S and 18S rRNA was visualized by EtBr staining as control. C Thin layer chromatogram of the F. fujikuroi wild-type strain IMI58289, the Ffvel1 knockout strain and the overexpression strain OE::Ffvel1 grown in 10 % ICI cultures for the indicated time. GA_4/7 and GA₃ were used as standards (for details see Experimental procedures).

Figure 5. Influence of nitrogen availability and ambient pH on gene expression in a Ffvel1 knock-out strain compared to the F. fujikuroi wild type IMI58289

A Expression of bik1-3, cps/ks, ggs2 and Ffvel1 in the wild type, the Ffvel1 and areA knockout mutants. The strains were grown for 72 h in 10 % ICI medium, washed and shifted into 0 % ICI or 100 % ICI medium, respectively, for 2 h. 28S and 18S rRNA was visualized by EtBr staining as control. B Expression of bik2, cps/ks, ggs2, areA, areB, nmrA and pacC in the wild type and the Ffvel1 knock-out mutant. The strains were grown for 72 h in 10 % ICI or 100 % ICI medium containing glutamine or NaNO₃, respectively. 28S and 18S rRNA was visualized by EtBr staining as control.

Figure 6. Multiple sequence alignment of FfLae1 with other characterized LaeA homologous sequences and restoration of secondary metabolism in A. nidulans ΔlaeA by Fflae1

A The alignment was generated using ClustalW. Putative HemK domains are marked in gray. Sequences aligned are: LaeA (AAQ95166) from A. nidulans, PcLaeA (ACD50375) from P. chrysogenum, and FfLae1 (FN548141) from F. fujikuroi. B Confirmation of heterologous complementation in A. nidulans by PCR. pFflae1-N-YFP: a plasmid containing the F. fujikuroi lae1 gene used for complementation of A. nidulans ΔlaeA strain RJW33.2, TJW97.6: RJW33.2 transformed with F. fujikuroi lae1, TJW97.11: RJW33.2 transformed with trpC marker gene. C Confirmation of heterologous complementation in A. nidulans by TLC. NOR was used as a standard for TLC. TJW97.6 produces similar amounts of NOR as TJW94.10, an A. nidulans laeA over-expressed strain. Negative control TJW97.11 (ΔlaeA) can produce a small amount of NOR.

Figure 7. Localization and interaction studies of FfVel1 and FfLae1 in F. fujikuroi

A Microscopic analysis of strains Ffvel1gfp and Fflae1gfp. The strains were grown for 3 days in 10 % ICI in the dark. Samples were stained with Hoechst 33342 and analyzed using an epifluorescence microscope as described in Experimental Procedures. B Microscopic analysis strains Ffvlyfp carrying Ffvel1 fused to the C-terminal part of YFP and Fflae1 fused to the N-terminal part of YFP. The strains were grown for 3 days in 10 % ICI in the dark. Samples were stained with Hoechst 33342 and analyzed using an epifluorescence microscope as described in Experimental Procedures.

Figure 8. Analysis of the Fflae1 knock-out strains compared to the F. fujikuroi wild type IMI58289 in regard to secondary metabolism and conidiation

A Thin layer chromatogram of the F. fujikuroi wild-type strain IMI58289, the Ffvel1 and Fflae1 knock-out strains as well as the overexpression strain OE::Ffvel1 grown in 10 % ICI cultures for 7 days. GA_4/7 and GA₃ were used as standards (for details see Experimental procedures). B Expression of bik1 and ggs2 in the wild type, the Ffvel1 and Fflae1 knock-out strains as well as the OE::Ffvel1 overexpression mutant. The strains were grown for 3 days in 10 % ICI medium. 28S and 18S rRNA was visualized by EtBr staining as control. C Photographs of the F. fujikuroi wild-type strain IMI58289, the Ffvel1 and Fflae1 knockout strains as well as the overexpression strain OE::Ffvel1 grown in 10 % ICI cultures for 3 days. D Spores produced by the F. fujikuroi wild-type strain IMI58289 and the Fflae1 knock-out strain grown for 10 days on V8 solidified media under constant light conditions. Experiment was carried out on triplicate; bars show standard deviations. E Expression of ggs2 and cps/ks in the wild type, the Ffvel1 and Fflae1 knock-out strains as well as in strains expressing ggs2 in the GA gene locus (ΔFfvel1+OE::ggs2^loc and ΔFflae1+OE::ggs2^loc) or ectopically (ΔFfvel1+OE::ggs2^{ect and} ΔFflae1+OE::ggs2^ect), respectively. The strains were grown for 3 days in 10 % ICI medium. 28S and 18S rRNA was visualized by EtBr staining as control.

Figure 9. Phenotypical and secondary metabolite analysis in the Ffvel2 knock-out strain compared to the F. fujikuroi wild type IMI58289

A Photographs of the F. fujikuroi wild type strain IMI58289, the Ffvel1 and Ffvel2 knockout strain and the overexpression strain OE::Ffvel1 grown on different solidified media for 10 days under constant dark conditions. V8 = vegetable juice; CM = complete medium; PDA = Potatoe Dextrose Agar; CD = Czapek Dox (for details see Experimental Procedures). B Spores produced by the F. fujikuroi wild type strain IMI58289, the Ffvel1 and Ffvel2 knock-out strains and the overexpression strain OE::Ffvel1 grown for 10 days on V8 solidified media under constant light conditions. Experiment was carried out in triplicate; bars show standard deviations. C Expression of selected gibberellin and bikaverin genes in the wild type and the Ffvel1 and Ffvel2 knock-out strains. The strains were grown for the indicated time in 10 % ICI medium. The northern blot was hybridized with the indicated probes. 28S and 18S rRNA was visualized by EtBr staining as control. D Photographs of the F. fujikuroi wild type strain IMI58289, the Ffvel1 and Ffvel2 knock-out strains grown in 10 % ICI cultures for 3 days. E Thin layer chromatogram of the F. fujikuroi wild type strain IMI58289 and the Ffvel1 and Ffvel2 knock-out strains grown in 10 % ICI cultures for the indicated time. GA₃ was used as standard (for details see Experimental procedures).